Compositions and assays to detect influenza virus A and B nucleic acids

ABSTRACT

Methods for detecting influenza virus A and influenza virus B nucleic acids in biological samples by using in vitro amplification and detection are disclosed. Compositions that are target-specific nucleic acid sequences and kits comprising target-specific nucleic acid oligomers for amplifying in vitro influenza virus A or influenza virus B nucleic acid and detecting amplified nucleic acid sequences are disclosed.

RELATED APPLICATION

This application is a divisional application of co-pending U.S.application Ser. No. 11/418,931, filed May 4, 2006, and claims benefitunder 35 U.S.C. 119(e) of provisional application No. 60/678,508, filedMay 6, 2005; each of which is incorporated by reference herein.

FIELD OF THE INVENTION

The field is detection of infectious agents, more specifically by usingcompositions and methods to detect influenza virus A and influenza virusB sequences by using in vitro nucleic acid amplification and probedetection.

BACKGROUND OF THE INVENTION

Influenza viruses (types A, B, and C) are members of theorthomyxoviridae family that cause influenza. Type A influenza virusesinfect birds and mammals, including humans, whereas types B and C infecthumans only. Influenza viruses are roughly spherical enveloped virusesof about 8-200 nm diameter that contain segmented negative sense genomicRNA. The envelope contains rigid structures that include hemagglutinin(HA) and neuraminidase (NA). Combinations of HA and NA subtypes, whichresult from genetic reassortment, are used to characterize viralisolates. Generally, influenza viral isolates are identified bynomenclature that includes type, location, isolate number, isolationyear, and HA and NA subtypes (e.g., “A/Sydney/7/97(H3N2)” refers to typeA, from Sydney, isolate 7, in 1997, with HA 3 and NA 2 subtypes). Minorgenetic changes that produce antigenic drift may cause influenzaepidemics, whereas genetic changes that result in a new HA or NA subtypeproduce antigenic shift that may cause a pandemic. Analysis of humaninfluenza virus A infections has shown that a few HA and NA combinationswere clinically significant in causing pandemics during the 1900s, i.e.,H1N1 in 1918, H2N2 in 1957, and H3N2 in 1968.

Influenza viruses that infect birds (e.g., chickens, ducks, pigeons) usecombinations of H5, H7 or H9 with any of N1 to N9. Since 1997, avianinfluenza viruses that have infected humans have included H5N1, H9N2,H7N2, and H7N7 viruses. Even limited human infections caused by an avianinfluenza virus raise concern for a potential pandemic, resulting inquarantines, and intentional destruction of large numbers of fowl, withaccompanying hardship. An avian influenza virus, or variant derivedtherefrom, that efficiently transfers by human-to-human contact couldcause a pandemic (Li et al., 2003, J. Virol. 77(12): 6988-6994).

Human influenza viruses produce highly contagious, acute respiratorydisease that results in significant morbidity and economic costs, withsignificant mortality among very young, elderly, and immuno-compromisedsubpopulations. Avian influenza infections in humans generally have ahigh mortality rate. A typical influenza virus infection in humans has ashort incubation period (1 to 2 days) and symptoms that last about aweek (e.g., abrupt onset of fever, sore throat, cough, headache,myalgia, malaise and anorexia), which may lead to pneumonia. Optimalprotection against infection requires annual inoculation with a vaccinethat includes a combination of types A and B of the most likely subtypesfor that year, based on global epidemiological surveillance. To beeffective in treatment, pharmaceuticals that block viral entry intocells or decrease viral release from infected cells must be administeredwithin 48 hrs of symptoms onset.

A variety of methods have been used to detect influenza virusesclinically. Viral culture in vitro (in monkey kidney cells) followed byvisual analysis and/or hemadsorption using microbiological methods candetect influenza viruses A and B in specimens (e.g., nasopharyngeal orthroat swab, nasal or bronchial wash, nasal aspirate, or sputum). Otherdetection tests include immunofluorescence assays (IFA), enzymeimmunoassays (EIA), and enzyme-linked immunosorbent assays (ELISA) thatuse antibodies specific to influenza virus antigens. Examples include asandwich microsphere-based IFA that uses influenza A- or B-specificmonoclonal antibodies and flow cytometry (Yan et al., 2004, J. Immunol.Methods 284(1-2): 27-38), monoclonal antibody-based EIA tests(DIRECTIGEN® FLU A and DIRECTIGEN® FLU A+B, Becton, Dickinson and Co.,Franklin Lakes, N.J., and QUICKVUE® Influenza Test, Quidel, San Diego,Calif.), and an immunoassay that produces a color change due toincreased thickness of molecular thin films when an immobilized antibodybinds an influenza A or B nucleoprotein (FLU OIA®, Biostar Inc.,Boulder, Colo.). Another chromagenic assay detects viral NA activity bysubstrate cleavage (ZSTAT FLU®, ZymeTx, Inc., Oklahoma City, Okla.).Assays are known that rely on reverse-transcriptase polymerase chainreactions (RT-PCR) to amplify influenza viral sequences to detectinfluenza A and B viruses (e.g., Templeton et al., 2004, J. Clin.Microbial 42(4):1564-69; Frisbie et al., 2004, J. Clin. Microbiol.42(3):1181-84; Boivin et al., 2004, J. Clin. Microbial, 42(1):45-51;Habib-Bein et al., 2003, J. Clin. Microbial 41(8):3597-3601; Li et al.,2001, J. Clin. Microbial 39(2):696-704; van Elden et al., 2001, J. aimMicrobiol. 39(1): 196-200; Fouchier et al., 2000, J. Clin. Microbiol.38(11):4096-101; Ellis et al., 1997, J. Clin. Microbial 35(8):2076-2082; PCT Nos. WO 2004 057021, WO 02 00884, WO 00 17391, and WO97/16570, EP Publ. No. 1 327 691 A2, U.S. Pat. No. 6,015,664, andPROFLU-1™ and HEXAPLEX™ tests, Prodesse, Milwaukee, Wis.). Serologydetects seroconversion associated with influenza virus A or B infectionsby detecting antibodies present in acute and convalescent sera frompatients with influenza symptoms. Detection methods have associatedadvantages and disadvantages related to sensitivity, specificity, assayand handling time, required equipment, and exposure of technicalpersonnel to infectious agents with related safety requirements forlaboratories and personnel. Generally, culture and serological testsrequire longer completion times (5 days to 2 weeks) with potentiallygreater exposure of technical personnel to infectious agents.Immunoassays are generally faster (30 min to 4 hrs) but often requiresubstantial sample handling and rely on subjective determination ofresults by technical personnel. PCR-based amplification assays may takeup to 2 days to complete and require specialized thermocyclingequipment. Hence a need remains for a test that provides sensitive,specific detection of influenza virus type A and type B in a relativelyshort time, with a minimum of exposure of technical personnel toinfectious agents, so that diagnosis is completed in sufficient time topermit effective therapeutic treatment of an infected person.

SUMMARY OF THE INVENTION

An embodiment disclosed herein is a composition that includes at leasttwo nucleic acid oligomers specific for influenza virus A made up ofsequences consisting of SEQ ID NO:3 to SEQ ID NO:18 and SEQ ID NO:21 toSEQ ID NO:31, or their completely complementary sequences, or DNAequivalents thereof. Preferred embodiments include nucleic acidoligomers in which at least one oligomer is selected from the sequencesconsisting of SEQ ID NO:7 to SEQ ID NO:18 and at least one oligomer isselected from the sequences consisting of SEQ ID NO:21 to SEQ ID NO:24.Another preferred embodiment also includes at least one oligomerselected from sequences consisting of SEQ ID NO:25 to SEQ ID NO:31. Inpreferred embodiments, at least one of the oligomers includes at leastone 2′-methoxy RNA group, whereas in other preferred embodiments atleast one of the oligomers includes at least one locked nucleic acid(LNA) residue at the 5′ end of the oligomer. In a preferred embodimentthat includes an oligomer selected from sequences consisting of SEQ IDNO:25 to SEQ ID NO:31, the oligomer also includes a detectable labeljoined directly or indirectly to the oligomer sequence. A preferredlabel is one that is detectable in a homogeneous assay system. Preferredembodiments of these compositions are kits that include at least two ofthe specified nucleic acid oligomers specific for influenza virus A.

Another embodiment disclosed herein is a composition that includes atleast two nucleic acid oligomers specific for influenza virus B made upof sequences consisting of SEQ ID NO:34 to SEQ ID NO:58, or theircompletely complementary sequences, or DNA equivalents thereof. Apreferred embodiment includes at least one oligomer selected from thesequences consisting of SEQ ID NO:38 to SEQ ID NO:43 and at least oneoligomer selected from the sequences consisting of SEQ ID NO:44 to SEQID NO:47. Another preferred embodiment also includes at least oneoligomer selected from sequences consisting of SEQ ID NO:48 to SEQ IDNO:58. In some preferred embodiments, at least one of the oligomersincludes at least one 2′-methoxy RNA group, whereas in other preferredembodiments, at least one of the oligomers includes at least one lockednucleic acid (LNA) residue at the 5′ end of the oligomer. In a preferredembodiment, the oligomer selected from sequences consisting of SEQ IDNO:48 to SEQ ID NO:58 includes a detectable label joined directly orindirectly to the oligomer sequence. Preferred embodiments include alabel that is detectable in a homogeneous assay system. Preferredembodiments of the compositions are kits that include at least two ofthe specified nucleic acid oligomers specific for influenza virus B.

Another embodiment is a method of detecting nucleic acid of influenzavirus A or influenza virus B in a sample, that includes the steps ofamplifying a target sequence in an influenza virus A nucleic acid orinfluenza virus B nucleic acid contained in a sample by using a nucleicacid polymerase in vitro to produce an amplified product undersubstantially isothermal conditions, wherein the target sequence ofinfluenza virus A is contained in SEQ ID NO:1, or its completecomplementary sequence, or RNA equivalents thereof, and wherein thetarget sequence of influenza virus B is contained in SEQ ID NO:32, orits complete complementary sequence, or RNA equivalents thereof, anddetecting the amplified product. In a preferred embodiment, the step ofamplifying the target sequence of influenza virus A uses at least oneoligomer selected from sequences consisting of SEQ ID NO:7 to SEQ IDNO:18 and one oligomer selected from sequences consisting of SEQ IDNO:21 to SEQ ID NO:24. In another embodiment, the step of amplifying thetarget sequence of influenza virus B uses at least one oligomer selectedfrom sequences consisting of SEQ ID NO:38 to SEQ ID NO:43 and oneoligomer selected from sequences consisting of SEQ ID NO:44 to SEQ IDNO:47. In another preferred embodiment, the detecting step uses at leastone probe selected from the sequences consisting of SEQ ID NO:25 to SEQID NO:31 to detect the amplified product of the target sequence ofinfluenza virus A, or at least one probe selected from the sequencesconsisting of SEQ ID NO:48 to SEQ ID NO:58 to detect the amplifiedproduct of the target sequence of influenza virus B. A preferredembodiment of the method also includes the steps of providing aninternal control oligomer, amplifying a target sequence contained in theinternal control oligomer, and detecting the amplified product made fromthe internal control oligomer, thereby indicating that the amplifyingand detecting steps of the method were properly performed. In anotherpreferred embodiment, the method also includes isolating an influenzavirus nucleic acid from the sample containing the influenza virus Anucleic acid or influenza virus B nucleic acid before the amplifyingstep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing signal detected (relative light units,RLU) for different amounts of influenza virus A target (250 to 5000copies per ml) in TMA-based assays performed manually (light bars) or byusing an automated system (dark bars).

FIG. 2 is a bar graph showing signal detected (relative light units,RLU) for different amounts of influenza virus B target (250 to 5000copies per ml) in TMA-based assays performed manually (light bars) or byusing an automated system (dark bars).

DETAILED DESCRIPTION OF THE INVENTION

Nucleic acid oligomer sequences are disclosed that may serve as primersfor amplification of influenza virus type A and influenza virus type Bnucleic acids present in samples by using methods of in vitro nucleicacid amplification, preferably by using a transcription-mediatedamplification reaction such as TMA or NASBA, and probes for detection ofthe amplified nucleic acid sequences. Detection probes hybridizespecifically to a portion of the amplified viral sequence, either aftercompletion of or during the amplification process. Some embodimentsdetect the amplified products by using a homogeneous detection methodthat detects, in a mixture, a labeled probe bound specifically to anamplified sequence (e.g., see Arnold et al., 1989, Clin. Chem.35:1588-1594; U.S. Pat. No. 5,658,737, Nelson et at, and U.S. Pat. Nos.5,118,801 and 5,312,728, Lizardi et al.). Embodiments of the methodsalso use oligonucleotide sequences that serve as capture probes forprocessing a sample to capture the target influenza virus nucleic acidand separate it from other sample components (U.S. Pat. Nos. 6,110,678,6,280,952 and 6,534,273, Weisburg et al.).

Methods disclosed herein detect influenza virus A and B nucleic acidspresent in samples derived from humans, preferably in nasopharyngeal orthroat swabs, nasal or bronchial washes, nasal aspirates, or sputum.Compositions disclosed herein include capture oligomers to separateinfluenza virus A or influenza virus B target nucleic acids from othercomponents in a sample, amplification oligomers to specifically amplifyselected nucleic acid sequences present in influenza virus genomicsequences, and nucleic acid probes for detecting the amplifiedsequences. Preferred embodiments include specific combinations ofoligomers to amplify and detect influenza virus type A and type Bsequences in assays that provide a detectable signal or response withinabout 45 min from beginning of a transcription-associated amplificationreaction.

The disclosed nucleic acid sequences and methods are useful foramplifying and detecting influenza virus type A and type B nucleic acidsfrom viral particles present in a sample in a relatively short time sothat diagnosis can be made during early stages of infection (e.g.,within 48 hr of symptoms) so that effective treatment can be initiated.The methods are useful for screening for individuals who have influenzavirus infections but who do not exhibit definitive symptoms,particularly for screening patients who have a higher risk of death orserious complications from influenza virus infections, e.g., young,elderly, or immunocompromised individuals. The methods are also usefulfor rapid screening of many samples, such as during an epidemic orpandemic, so that appropriate public health responses can be initiated.The methods are useful because they minimize the risk of exposure oflaboratory personnel to infectious agents, such as an avian influenzavirus related to influenza virus type A or type B that has becomeinfectious to humans. Thus, the methods and compositions disclosedherein respond to a need for rapid, sensitive, and specific testing ofclinical samples that may contain influenza virus A or virus B.

A “sample” or “specimen”, including “biological” or “clinical” samples,refers to a tissue or material derived from a living or dead human oranimal which may contain an influenza virus target nucleic acid,including, for example, nasopharyngeal or throat swabs, nasal orbronchial washes, nasal aspirates, sputum, other respiratory tissue orexudates, or biopsy tissue including lymph nodes. A sample may betreated to physically or mechanically disrupt tissue or cell structureto release intracellular nucleic acids into a solution which may containenzymes, buffers, salts, detergents and the like, to prepare the samplefor analysis.

“Nucleic acid” refers to a multimeric compound comprising nucleosides ornucleoside analogs which have nitrogenous heterocyclic bases or baseanalogs linked together to form a polynucleotide, including conventionalRNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. Anucleic acid “backbone” may be made up of a variety of linkages,including one or more of sugar-phosphodiester linkages, peptide-nucleicacid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose, orsimilar compounds with substitutions, e.g., 2′ methoxy or 2′ halidesubstitutions. Nitrogenous bases may be conventional bases (A, G, C, T,U), analogs thereof (e.g., inosine or others; see The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11^(1h) ed., 1992),derivatives of purines or pyrimidines (e.g., N⁴-methyl deoxygaunosine,deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases withsubstituent groups at the 5 or 6 position, purine bases with asubstituent at the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; U.S. Pat. No.5,378,825 and PCT No. WO 93/13121). Nucleic acids may include one ormore “abasic” residues where the backbone includes no nitrogenous basefor position(s) of the polymer (U.S. Pat. No. 5,585,481). A nucleic acidmay comprise only conventional RNA or DNA sugars, bases and linkages, ormay include both conventional components and substitutions (e.g.,conventional bases with 2′ methoxy linkages, or polymers containing bothconventional bases and one or more base analogs). Nucleic acid includes“locked nucleic acid” (LNA), an analogue containing one or more LNAnucleotide monomers with a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhance hybridization affinitytoward complementary RNA and DNA sequences (Vester and Wengel, 2004,Biochemistry 43(42):13233-41). Embodiments of oligomers that may affectstability of a hybridization complex include PNA oligomers, oligomersthat include 2′-methoxy or 2′-fluoro substituted RNA, or oligomers thataffect the overall charge, charge density, or steric associations of ahybridization complex, including oligomers that contain charged linkages(e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates).

An “oligomer” or “oligonucleotide” refers to a nucleic acid of generallyless than 1,000 nucleotides (nt), including those in a size range havinga lower limit of about 2 to 5 nt and an upper limit of about 500 to 900nt. Some preferred embodiments are oligomers in a size range with alower limit of about 5 to 15 nt and an upper limit of about 50 to 600nt, and other preferred embodiments are in a size range with a lowerlimit of about 10 to 20 nt and an upper limit of about 22 to 100 nt.Oligomers may be purified from naturally occurring sources, butpreferably are synthesized by using any well known enzymatic or chemicalmethod. Oligomers may be referred to by a functional name (e.g., captureprobe, primer or promoter primer) but those skilled in the art willunderstand that such terms refer to oligomers.

A “capture probe”, “capture oligonucleotide”, or “capture oligomer”refers to a nucleic acid oligomer that specifically hybridizes to atarget sequence in a target nucleic acid by to standard base pairing andjoins to a binding partner on an immobilized probe to capture the targetnucleic acid to a support. A preferred embodiment of a capture oligomerincludes two binding regions: a sequence-binding region (i.e.,target-specific portion) and an immobilized probe-binding region,usually on the same oligomer, although the two regions may be present ontwo different oligomers joined together by one or more linkers.

An “immobilized probe”, “immobilized oligomer” or “immobilized nucleicacid” refers to a nucleic acid binding partner that joins a captureoligomer to a support, directly or indirectly. An immobilized probejoined to a support facilitates separation of a capture probe boundtarget from unbound material in a sample. Any support may be used, e.g.,matrices or particles free in solution, which may be made of any of avariety of materials, e.g., nylon, nitrocellulose, glass, polyacrylate,mixed polymers, polystyrene, silane polypropylene, or metal. Preferredembodiments use a support that is magnetically attractable particles,e.g., monodisperse paramagnetic beads (uniform size ±5%) to which animmobilized probe is joined directly (e.g., via covalent linkage,chelation, or ionic interaction) or indirectly (e.g., via a linker),where the joining is stable during nucleic acid hybridizationconditions.

“Separating” or “purifying” refers to removing one or more components ofa sample from one or more other sample components, e.g., removing somenucleic acids from a generally aqueous solution that may also containproteins, carbohydrates, lipids, or other nucleic acids. In preferredembodiments, a separating or purifying step removes the target nucleicacid from at least about 70%, more preferably at least about 90% and,even more preferably, at least about 95% of the other sample components.

An “amplification oligonucleotide” or “amplification oligomer” refers toan oligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction,e.g., serving as a primer or and promoter-primer. Preferredamplification oligomers contain at least about 10 contiguous bases, andmore preferably at least 12 contiguous bases, that are complementary toa region of the target nucleic acid sequence or its complementarystrand. The contiguous bases are preferably at least about 80%, morepreferably at least about 90%, and most preferably completelycomplementary to the target sequence to which the amplification oligomerbinds. Preferred amplification oligomers are about 10 to about 60 baseslong and optionally may include modified nucleotides. A “primer” refersto an oligomer that hybridizes to a template nucleic acid and has a 3′end that is extended by polymerization. A primer may be optionallymodified, e.g., by including a 5′ region that is non-complementary tothe target sequence. A primer modified with a 5′ promoter sequence isreferred to as a “promoter-primer.” A person of ordinary skill in theart of molecular biology or biochemistry will understand that anoligomer that can function as a primer can be modified to include a 5′promoter sequence and then function as a promoter-primer, and,similarly, any promoter-primer can serve as a primer with or without its5′ promoter sequence.

“Nucleic acid amplification” refers to any well known in vitro procedurethat produces multiple copies of a target nucleic acid sequence, or itscomplementary sequence, or fragments thereof (i.e., an amplifiedsequence containing less than the complete target nucleic acid).Examples of well known nucleic acid amplification procedures includetranscription associated methods, such as transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA)and others (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990,5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification(e.g., U.S. Pat. No. 4,786,600), the polymerase chain reaction (PCR)(e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chainreaction (LCR) (e.g., EP Pat. App. 0320308) and strand-displacementamplification (SDA) (e.g., U.S. Pat. No. 5,422,252). Replicase-mediatedamplification uses self-replicating RNA molecules, and a replicase suchas QB-replicase. PCR amplification uses DNA polymerase, primers, andthermal cycling steps to synthesize multiple copies of the twocomplementary strands of DNA or cDNA. LCR amplification uses at leastfour separate oligonucleotides to amplify a target and its complementarystrand by using multiple cycles of hybridization, ligation, anddenaturation. SDA uses a primer that contains a recognition site for arestriction endonuclease that will nick one strand of a hemimodified DNAduplex that includes the target sequence, followed by amplification in aseries of primer extension and strand displacement steps. Preferredembodiments use a transcription associated amplification, such as TMA orNASBA, but it will be apparent to persons of ordinary skill in the artthat oligomers disclosed herein may be readily used as primers in otheramplification methods. Briefly, transcription associated amplificationuses a DNA polymerase, an RNA polymerase, deoxyribonucleosidetriphosphates, ribonucleoside triphosphates, a promoter-containingoligonucleotide, and optionally may include other oligonucleotides, toultimately produce multiple RNA transcripts from a nucleic acid template(described in detail in U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacianet al., U.S. Pat. No. 5,437,990, Burg et al., PCT Nos. WO 88/01302 andWO 88/10315, Gingeras et al., U.S. Pat. No. 5,130,238, Malek et al.,U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al, PCT No. WO94/03472, McDonough at al., PCT No. WO 95/03430, and Ryder et al).Preferred methods that use TMA as described in detail previously (U.S.Pat. Nos. 5,399,491 and 5,554,516).

“Detection probe” refers to a nucleic acid oligomer that hybridizesspecifically to a target sequence, including an amplified sequence,under conditions that promote nucleic acid hybridization, for detectionof the target nucleic acid. Detection may either be direct (i.e., probehybridized directly to the target) or indirect (i.e., a probe hybridizedto an intermediate structure that links the probe to the target). Aprobe's target sequence generally refers to the specific sequence withina larger sequence which the probe hybridizes specifically. A detectionprobe may include target-specific sequences and other sequences orstructures that contribute to the probe's three-dimensional structure,depending on whether the target sequence is present (e.g., U.S. Pat.Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412).

“Label” refers to a moiety or compound joined directly or indirectly toa probe that is detected or leads to a detectable signal. Direct joiningmay use covalent bonds or non-covalent interactions (e.g., hydrogenbonding, hydrophobic or ionic interactions, and chelate or coordinationcomplex formation) whereas indirect joining may use a bridging moiety orlinker (e.g., via an antibody or additional oligonucleotide(s), whichamplify a detectable signal. Any detectable moiety may be used, e.g.,radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate,reactive group, chromophore such as a dye or particle (e.g., latex ormetal bead) that imparts a detectable color, luminescent compound (e.g.bioluminescent, phosphorescent or chemiluminescent compound), andfluorescent compound. Preferred embodiments include a “homogeneousdetectable label” that is detectable in a homogeneous system in whichbound labeled probe in a mixture exhibits a detectable change comparedto unbound labeled probe, which allows the label to be detected withoutphysically removing hybridized from unhybridized labeled probe (e.g.,U.S. Pat. Nos. 5,283,174, 5,656,207 and 5,658,737). Preferredhomogeneous detectable labels include chemiluminescent compounds, morepreferably acridinium ester (“AE”) compounds, such as standard AE or AEderivatives which are well known (U.S. Pat. Nos. 5,656,207, 5,658,737,and 5,639,604). Methods of synthesizing labels, attaching labels tonucleic acid, and detecting signals from labels are well known (e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) atChapt. 10, and US Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174,and 4,581,333, and EP Pat. App. 0 747 706). Preferred methods of linkingan AE compound to a nucleic acid are known (e.g., U.S. Pat. No.5,585,481 and U.S. Pat. No. 5,639,604, see column 10, line 6 to column11, line 3, and Example 8). Preferred AE labeling positions are aprobe's central region and near a region of A/T base pairs, at a probe's3′ or 5′ terminus, or at or near a mismatch site with a known sequencethat is the probe should not detect compared to the desired targetsequence.

Sequences are “sufficiently complementary” if they allow stablehybridization of two nucleic acid sequences, e.g., probe and targetsequences, although the sequences are not completely complementary. Thatis, a “sufficiently complementary” sequence that hybridizes to anothersequence by hydrogen bonding between a subset series of complementarynucleotides by using standard base pairing (e.g., G:C, A:T or A:U),although the two sequences may contain one or more residues (includingabasic positions) that are not complementary so long as the entiresequences in appropriate hybridization conditions to form a stablehybridization complex. Sufficiently complementary sequences arepreferably at least about 80%, more preferably at least about 90%, andmost preferably completely complementary in the sequences that hybridizetogether. Appropriate hybridization conditions are well known to thoseskilled in the art, can be predicted based on sequence composition, orcan be determined empirically by using routine testing (e.g., Sambrooket al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed. at§§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly§§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

“Consisting essentially of” means that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the compositions and methods describedherein may be included in those compositions or methods. Suchcharacteristics include the ability to detect an influenza virus A orinfluenza virus B nucleic acid sequence present in a sample withspecificity that distinguishes the influenza virus nucleic acid from atleast 50 other known respiratory pathogens, preferably at a sensitivitythat detects at least 1.7 to 2.7 log copies of the influenza virus,within about 45 min from the beginning of an amplification reaction thatmakes amplified viral sequences that are detected.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. General definitions may be found in technical booksrelevant to the art of molecular biology, e.g., Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.) or The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.). Unlessmentioned otherwise, techniques employed or contemplated herein arestandard methodologies well known to one of ordinary skill in the art.The examples included herein illustrate some preferred embodiments.

Compositions that include nucleic acid oligomers that function in targetcapture, amplification, and detection of nucleic acids and methods fordetecting human influenza virus A (“FluA”) and influenza virus B(“FluB”) nucleic acid present in a biological sample are disclosedherein. To select target sequences appropriate for use in the tests todetect influenza virus A, known influenza virus A RNA or DNA sequencesthat encode a matrix protein (MP1), including partial or complementarysequences (available at publicly accessible databases, e.g., GenBank),were aligned by matching regions of identical or similar sequences andcompared. To select target sequences appropriate for use in the tests todetect influenza virus B, known influenza virus B RNA or DNA sequencesthat encode a non-structural protein (NS1), including partial orcomplementary sequences (available at publicly accessible databases,e.g., GenBank) were aligned by matching regions of identical or similarsequences and compared. Although sequence comparisons may be facilitatedby use of computer-performed algorithms, one of ordinary skill canperform the comparisons manually and visually. Portions of sequences foreach viral target that contained relatively few sequence changes betweenthe compared individual viral sequences were chosen as a basis fordesigning synthetic oligomers for use in the methods described herein.Oligonucleotide sequences for detecting the FluA target are shown inTable 1, and sequences for detecting the FluB target are shown in Table2. In both tables, a preferred function is included for each sequence,and for sequences identified as promoter primers as the preferredfunction, the sequences include a 5′ T7 bacteriophage promoter sequence(underlined, consisting of SEQ ID NO:19 or SEQ ID NO:20) from which a T7RNA polymerase can initiate transcription under appropriate conditions.Those skilled in the art will appreciate that another 5′ promotersequence may be substituted for the underlined T7 promoter sequence,which would then function with the appropriate RNA polymerase for thechosen other promoter sequence, to make an equivalent promoter primeroligonucleotide. Oligomers having the same target-specific sequences asthe promoter primers but without the promoter sequence are also shown(SEQ ID Nos. 13-18 and 41-43) which are capable of functioning asprimers in amplification systems that do not use promoter primers. Thoseskilled in the art will recognize that oligomers identified as having apreferred function in target capture have target-specific portions(shown in SEQ ID Nos. 3, 4, 36, and 37) and optionally include tailportions (T₃A₃₀ shown in SEQ ID Nos. 5, 6, 34, and 35) which may bedeleted or substituted with other sequences or binding moieties.

TABLE 1 Oligomer Sequences Specific for Influenza virus A DetectionAssays Preferred SEQ ID Sequence Function 3 gaggcccatgcaactggcaagtgcactarget capture 4 gaatccacaatatcaagtgcaagatccc target capture 5gaggcccatgcaactggcaagtgcacttt(a)₃₀ target capture 6gaatccacaatatcaagtgcaagatcccttt(a)₃₀ target capture 7aatttatacgactcactatagggagaagggcattttggacaaakcgt promoter primer ct 8aatttatacgactcactatagggagaagggcattttggacaaagcgt promoter primer ct 9aatttaatacgactcactatagggagaagggcattttggacaaakcg promoter primer tc 10aatttaatacgactcactatagggagaagggcattttggacaaagcg promoter primer tc 11aatttaatacgactcactatagggagaagggcattttggacaaakcgt promoter primer 12aatttaatacgactcactatagggagaagggcattttggacaaagcgt promoter primer 21catggartggctaaagacaa primer 22 catggartggctaaagacaaga primer 23gtrttcacgctcaccgtgc primer 24 catggartggctaaagacaagacc primer 25caccgugcccagugagc probe 26 gcccagugagcgagga probe 27 cgaggacugcagcguagprobe 28 ggcucgugcccagugagcgagggagcc probe 29gugcccagugagcgaggacugcggcac probe 30 ggcuccagugagcgaggacugcagagcc probe31 ggcucugagcgaggacugcagcgagcc probe

TABLE 2 Oligomer Sequences Specific for Influenza virus B DetectionAssays Preferred SEQ ID Sequence Function 34gucuugaccaggguagucaaggttt(a)₃₀ target capture 35ggcucaaacccuucaauuccttt(a)₃₀ target capture 38aatttaatacgactcactatagggagacggtgctcttgaccaaattgg promoter primer 39aatttaatacgactcactatagggagacggtgctcttgaccaaatt promoter primer 40aatttaatacgactcactatagggagacggtgctcttgaccaaattg promoter primer 44tcctcaactcactcttcga primer 45 tcctcaactcactcttcgagcg primer 46tcctcaactcactcttcgagc primer 47 gaaggacattcaaagcc primer 48gccaauucgagcagcug probe 49 gagcagcugaaacugcg probe 50gcagcugaaacugcggugg probe 51 cgcacgcagcugaaacugcggugcg probe 52ccagcgccaauucgagcagcugg probe 53 cgguggcugaaacugcgguggcaccg probe 54ggcucuucgagcagcugaaacuggagcc probe 55 ggcucucgagcagcugaaacuggagcc probe56 ggcucauucgagcagcugaaacugugagcc probe 57ggcucguucgagcagcugaaacugcgagcc probe 58 cgcagucgagcagcugaaacugcg probe

Although sequences are shown in Tables 1 and 2 as DNA, RNA or mixedDNA/RNA sequences, the sequences are meant to include the correspondingDNA or RNA sequences, and their completely complementary DNA or RNAsequences. Preferred embodiments of oligomers may include one or moremodified residues affecting the backbone structure (e.g., 2′-methoxysubstituted RNA groups), or one or more LNA monomers, preferably at 5′residues of a primer oligomer, or may include a non-nucleotide linker toattach a label to the oligomer. For example, oligomers that function asprobes for RNA targets may be synthesized with 2′-methoxy substitutedRNA groups to promote more stable hybridization between probe and targetsequences. Embodiments include oligomers of SEQ ID Nos. 25-27synthesized with 2′-methoxy substituted RNA groups and having anon-nucleotide linker (as described in U.S. Pat. No. 5,585,481) betweenresidues 6 and 7 of SEQ ID NO:25, between residues 7 and 8 of SEQ IDNos. 26 and 50, between residues 8 and 9 of SEQ ID Nos. 26 and 27, andbetween residues 9 and 10 of SEQ ID Nos. 48 and 49. Other embodimentsinclude oligomers of SEQ ID Nos. 44 and 45 synthesized with LNA at 5′residues 1 to 3, and an oligomer of SEQ ID NO:46 synthesized with LNA at5′ residues 1 to 4.

Preferred embodiments of target capture oligomers include atarget-specific sequence that binds specifically to the FluA or FluBtarget nucleic acid and a covalently linked “tail” sequence (T₃A₃₀ inSEQ ID Nos. 5, 6, 34 and 35) used in capturing the hybridization complexcontaining the target nucleic acid to an immobilized sequence on a solidsupport. Preferred embodiments of capture oligomers include at least one2′ methoxy linkage. Embodiments of capture oligomers may include thetarget-specific sequence that binds to a FluA or FluB genomic sequenceattached to another binding moiety, e.g., a biotinylated sequence thatbinds specifically to immobilized avidin or streptavidin. The tailsequence or binding moiety binds to an immobilized probe (e.g.,complementary sequence or avidin) to capture the hybridized target andseparate it from other sample components by separating the solid supportfrom the mixture.

Primer sequences, including promoter primer sequences, bind specificallyto the target nucleic acid or its complementary sequence and may containadditional sequences that are not target-specific, e.g., the promotersequence in a promoter primer. A target-specific sequence, with orwithout an attached promoter sequence, may serve as an amplificationoligomer in a variety of in vitro amplification processes. Embodimentsof the FluA and FluB assays may use amplification methods that requiremultiple cycling reaction temperatures, such as PCR (U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159), or may be substantially isothermalas in transcription associated amplification methods, such as TMA orNASBA (e.g., U.S. Pat. Nos. 5,399,491, 5,480,784, 5,824,518, 5,888,779,5,786,183, 5,437,990, 5,130,238, 4,868,105, and 5,124,246, and PCT Nos.WO 8801302 and WO 8810315). Preferred embodiments of the FluA and FluBassays use PCR-based or TMA-based amplification systems that aredetected during the amplification process (i.e., real time detection) byincluding probes that emit distinguishable fluorescent signals when theprobe is bound to the intended target sequence made during theamplification process. Preferred probes for real time detection includethose referred to as “molecular beacon” or “molecular switch” probes(e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al., U.S. Pat.Nos. 5,925,517 and 6,150,097, Tyagi et al., Giesendorf et al., 1998,Clin. Chem. 44(3):482-6) and “molecular torch” probes (e.g., U.S. Pat.Nos. 6,835,542 and 6,849,412, Becker et al.). Generally, such probesinclude a reporter dye attached to one end of the probe oligomer (e.g.,FAM™, TET™, JOE™, VIC™) and a quencher compound (e.g., TAMRA™ ornon-fluorescent quencher) attached to the other end of the probeoligomer, and signal production depends on whether the two ends withtheir attached compounds are in close proximity or separated.

The assay to detect influenza virus in a sample includes the steps ofamplifying a target region in the target influenza virus nucleic acidcontained in a sample by using amplification oligomers or primersspecific for the intended target region, and detecting the amplifiednucleic acid by hybridizing it to a probe sequence. Preferred assays usea transcription-associated amplification reaction and detection isduring the amplification reaction. For detection, the amplified nucleicacid may be labeled and bound to an unlabeled probe, but preferredembodiments bind a labeled probe to the amplified nucleic acid. Apreferred embodiment for real-time detection uses a labeled probe thatis detected in a homogeneous system.

Generally, the target influenza virus nucleic acid is separated fromother sample components before the amplification step. This may be doneby capturing the influenza virus nucleic acid by using a target-specificcapture oligomer that binds specifically to the target influenza virusnucleic acid, or by using non-specific methods of purifying nucleic acidfrom a sample (e.g., U.S. Pat. Nos. 5,234,809, 5,705,628, 6,534,262 and6,939,672). Preferred embodiments use uses a target-specific captureoligomer in a capturing step (U.S. Pat. Nos. 6,110,678, 6,280,952 and6,534,273). Embodiments of capture probes include those specific forinfluenza virus A nucleic acid (SEQ ID Nos. 3 to 6), and those specificfor influenza virus B nucleic acid (SEQ ID Nos. 34 to 37). Embodimentsof SEQ ID Nos. 5, 6, 34, and 35 include a dT₃A₃₀ tail portion forhybridization to a complementary immobilized sequence, whereasembodiments of SEQ ID Nos. 3, 4, 36 and 37 are used in conjunction withanother ligand that is a member of a binding pair (e.g., biotinylatedDNA to bind to immobilized avidin or streptavidin). The complex thecapture probe, its target influenza virus nucleic acid, and animmobilized binding partner or probe facilitate separation of theinfluenza virus nucleic acid from other sample components, and optionalwashing steps may be used to further purify the captured viral nucleicacid.

Amplifying the influenza virus target region using two primers may beaccomplished using a variety of known nucleic acid amplificationreactions, but preferably uses a transcription-associated amplificationreaction, such as TMA (described in detail in US Pat. Nos. 5,399,491 and5,554,516). A TMA-based assay produces many RNA transcripts (amplicons)from a single copy of target nucleic acid, and the amplicons aredetected to indicate the presence of the target influenza virus in thesample. Briefly, in TMA-based assays, a promoter-primer hybridizesspecifically to the target sequence and reverse transcriptase (RT) thatincludes RnaseH activity creates a first strand cDNA by extension fromthe 3′ end of the promoter-primer and digests the template strand. ThecDNA is then bound by a second primer and a new strand of DNA issynthesized from the end of the second primer using RT to create adouble-stranded DNA (dsDNA) containing a functional promoter sequence.RNA polymerase specific for that promoter binds to the promoter sequenceand multiple RNA transcripts are produced, which each can act as atemplate for additional sequence replication using the same steps usedfor the initial template. Thus, large amounts of single-strandedamplified product are made using substantially isothermal reactionconditions.

Another embodiment of the influenza virus assay uses PCR amplification(U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, Mullis et al.) toproduce multiple DNA strands by using thermocycling reactions thatseparate dsDNA and primers specific for portions of the separatedstrands to make additional dsDNA molecules by using a DNA polymerase.Well known variations of the basic PCR method may also be used, e.g.,reverse-transcriptase PCR that uses RT to produce a cDNA from an RNAtemplate, and then the DNA is amplified by PCR cycles, or PCR coupledwith real-time detection, both of which are sometimes referred to asRT-PCR (e.g., TaqMan One-Step RT-PCR kits, Applied Biosystems, Inc.,Foster City, Calif.).

Embodiments of amplification oligomers specific for influenza virus Anucleic acid include promoter primers of SEQ ID Nos. 7 to 12,target-specific sequences of SEQ ID Nos. 13 to 18 which are contained inthe promoter primers, and SEQ ID Nos. 19 to 24. A preferred compositionincludes a mixture of oligomers of SEQ ID NO:7 and SEQ ID NO:21, whereSEQ ID NO:21 has LNA residues at positions 1 to 3, which composition isuseful for TMA-based amplification of the influenza virus A targetregion of SEQ ID NO:1.

Embodiments of amplification oligomers specific for influenza virus Bnucleic acid include promoter primers of SEQ ID Nos. 38 to 40,target-specific sequences of SEQ ID Nos. 41 to 43 which are contained inthe promoter primers, and SEQ ID Nos. 44 to 47. A preferred compositionincludes a mixture of oligomers of SEQ ID NO:39 and SEQ ID NO:44, whereSEQ ID NO:44 includes LNA residues at positions 1 to 3, whichcomposition is useful for TMA-based amplification of the influenza virusB target region of SEQ ID NO:32.

The methods for detecting influenza virus nucleic acid include adetecting step that uses at least one probe that binds specifically tothe amplified influenza virus product (RNA or DNA amplicons).Preferably, the probe is labeled and produces a signal detected in ahomogeneous system, i.e., without separation of bound probe from unboundprobe. Preferred probes are labeled with an acridinium ester (AE)compound from which a chemiluminescent signal is produced and detectedin a homogeneous system (substantially as described in detail in U.S.Pat. Nos. 5,283,174, 5,656,744, and 5,658,737). Other preferred probesare labeled with a fluorescent compound which emits a detectable signalonly when the probe is bound to its target, e.g., molecular switch,beacon, or torch probes. Preferred probes for specific detection ofinfluenza virus A sequences include oligomers of SEQ ID Nos. 25 to 27,preferably AE-labeled between residues 6 and 7 for SEQ ID NO:25, betweenresidues 7 and 8 or 8 and 9 for SEQ ID NO:26, and between residues 8 and9 for SEQ ID NO:27. Other preferred probes for specific detection ofinfluenza virus A sequences include fluorescent compound-labeledoligomers of SEQ ID Nos. 28 to 31. Preferred probes for specificdetection of influenza virus B sequences include oligomers of SEQ IDNos. 48 to 50, preferably AE-labeled between residues 9 and 10 for SEQID Nos. 48 and 49, and between residues 7 and 8 for SEQ ID NO:50. Otherpreferred probes for specific detection of influenza virus A sequencesinclude fluorescent compound-labeled oligomers of SEQ ID Nos. 51 to 58.

Preferred embodiments of assays for detection of influenza virus A or Bnucleic acid include an internal control (IC) nucleic acid that isamplified and detected by using IC-specific primers and probe in thesame reaction mixtures used for influenza virus nucleic acidamplification and detection. Amplification and detection the IC-specificsequence demonstrates that assay reagents and conditions were properlyused even when no influenza virus-specific signal is detected for atested sample (i.e., negative samples). The IC may be used as aninternal calibrator for the assay that provides a quantitative result. Apreferred IC embodiment is a randomized sequence derived from anaturally occurring source that is not an influenza virus (e.g., HIV).An preferred IC is SEQ ID NO:65 or its RNA transcript, and preferredembodiments of primers for amplification of this IC include those of SEQID Nos. 61, 62, 64, 66, and 67. Probes for detection of IC ampliconsinclude any oligomer of at least ten residues that hybridizesspecifically to a contiguous sequence contained in SEQ ID NO:65 or itscomplement (DNA or RNA) under assay conditions described herein. Apreferred IC-specific probe is exemplified by an oligomer of SEQ IDNO:63 labeled with a fluorescent compound at one end and a quencher atthe other end. In preferred embodiments that include an IC in an assay,the IC is treated throughout the assay similar to the intended analyte.For example, when a target capture step is used for purification of theinfluenza virus nucleic acid target in a sample, the target capture stepincludes a capture oligomer specific for the IC to purify the IC from amixture that includes the target influenza virus nucleic acid and othersample components. Preferred embodiments of capture oligomers specificfor the IC of SEQ ID NO:65 include those of SEQ ID Nos. 59 and 60.

In general, methods used to demonstrate amplification and detection ofinfluenza virus A or B nucleic acid by using the compositions describedherein involved the following steps. Influenza viral RNA was separatedfrom other sample components by using a method that attaches the targetinfluenza virus nucleic acid to a solid support that is separated fromother sample components. In preferred embodiments, viral RNA wasseparated from other sample components by using a target-capture systemthat included a target-specific capture probe for is the influenza viralanalyte (e.g., using methods steps described in U.S. Pat. Nos.6,110,678, 6,280,952 and 6,534,273), or a non-specific method forseparation of nucleic acids was used (U.S. Pat. No. 5,234,809).Non-specific separation of viral RNA from other sample components wasperformed by adhering nucleic acids reversibly to a solid support,followed by washing and elution of the adhered nucleic acids into asubstantially aqueous solution (e.g., using a QIAAMP™ Viral RNA Minikit, Qiagen Inc.). Isolated influenza virus nucleic acid was amplifiedfor specific target sequences contained the genome by using TMA or PCRamplification, and the amplification products were detected aftercompletion of the amplification reaction or during amplification (i.e.,real-time detection). For real-time detection, a fluorophore-labeledprobe (e.g., molecular beacon) was used that emits a detectable signalonly when the probe is hybridized to its target sequence, andfluorescence was detected using standard fluorometry. Generally, assaysdetected two different probes (with different 5′ fluorophores): aninfluenza virus-specific probe and an IC-specific probe. Fluorescencewas detected by using a system that incubates the reactions and detectsfluorescence at different wavelengths at time intervals during thereaction (e.g., DNA Engine OPTICON™ 2 system or CHROMO4™ Real-Time PCRDetector, Bio-Rad Laboratories, Inc., Hercules, Calif.). Real-timedetected fluorescent signals in each channel were analyzed usingstandard methods. For example, detected signals were normalized togenerate a best-fit curve to the data points for each reaction (relativefluorescence vs. time) and results were reported as the time ofemergence when the signal met or exceeded a pre-set level.

For comparison with TMA-based assays, real-time reverse-transcriptasePCR-based assays (RT-PCR) were performed by using 0.9 pmol/μl of primers(SEQ ID NO:68 and SEQ ID NO:69 for influenza virus A, or SEQ ID NO:71and SEQ ID NO:72 for influenza virus B) and 0.2 pmol/μl of probe (SEQ IDNO:70 for influenza virus A and SEQ ID NO:73 for influenza virus B) in a50 μl reaction that included standard PCR reaction components (providedby TAQMAN® One-Step RT-PCR Master Mix Reagents Kit, Applied Biosystems,Inc.). Incubation was performed using: 48° C. for 30 min, 95° C. for 10min, then 45 cycles of 95° C. for 15 sec and cooling, and finally 60° C.for 1 min. Amplification and detection of the molecular beacon probehybridized to its target amplified product were performed by using anopen channel system (CHROMO4™, Bio-Rad Laboratories, Inc.) for real-timefluorescence detection, with fluorescent signal readings taken at eachof the 45 cycles. Real-time fluorescence signals were analyzed anddetection of the analytes calculated from the fluorescence emergencecurves by using standard methods.

Real-time TMA-based assays typically were performed in reaction mixturethat contained the analyte nucleic acid, amplification reagent (APTIMA™reagent, Gen-Probe Incorporated, San Diego, Calif.), a T7 promoterprimer that included (9 pmol/reaction), a second primer without apromoter (15 pmol/reaction), and a molecular beacon probe (0.2-0.3pmol/reaction) for amplicon detection, in a 40 μl reaction (in a well ofa standard 96-well plate, covered with a layer of inert oil or sealingdevice to prevent evaporation). The mixture of target nucleic acid,primers, and probe was incubated at 6° C. for 10 min, cooled at 42° C.for 5 min, and then enzyme reagent containing RT and T7 RNA polymerasewas added, the mixture was mixed (e.g., 30 sec vortex) and thenincubated at 42° C. for 75-100 min for isothermal amplification duringwhich detection of fluorescence was performed every 3 sec. Amplificationand detection steps were performed using an incubation and open channelfluorimeter (CHROMO4™, Bio-Rad Laboratories, Inc.) for real-timetwo-color fluorescence detection. Generally, the assays included an IC,as described above, i.e., a reaction mixture included primers and probefor the target influenza virus nucleic acid and IC-specific primers andprobe, each probe labeled with a separately detectable 5′ fluorophore.Real-time fluorescence signals were analyzed and a detection signal(time of emergence) was calculated. Time of emergence was calculated,e.g., by using a method that analyzes the detected signals (relativefluorescence units or RFU) relative to the signal detection times(RFU(t) data points) to determine a time of emergence (“T-time”), whichis the time at which a RFU(t) data point reaches a predefined thresholdvalue (described in detail in U.S. application 60/659,874, Scalese etal., filed Mar. 10, 2005). Briefly, RFU(t) data is treated to subtractbackground signal (“noise” level) and curves (RFU vs time) arenormalized to optimize curve fit for data between predetermined minimumand maximum points. In general, samples that contain a higher analyteconcentration result in a steeper curve slope and an earlier time ofemergence. In the examples described herein, samples often containedknown amounts of the target influenza virus nucleic acid (influenzavirus A or B RNA, expressed as “log copies” per reaction) and theaverage (mean) time of emergence (average Ttime) was determined forreplicate tests performed identically. Average times of emergence werecompared to determine the relative efficiencies of the different assayconditions, e.g., to compare for a single known amount of analyte, thetime of emergence detected by using a PCR-based assay compared to usinga TMA-based assay. For TMA-based assays that included an IC, the IC wasa known amount of RNA transcripts made in vitro from SEQ ID NO:65 thatwas amplified by using IC-specific primers in the same reaction as foramplification of the target influenza virus nucleic acid. Generally, forinfluenza A virus assays, IC-specific primers were SEQ ID NO:61 (0.5pmol/reaction) and SEQ ID NO:62 (15 pmol/reaction), and for influenzavirus B to assays, IC-specific primers were SEQ ID NO:64 (0.5pmol/reaction) and SEQ ID NO:62 (15 pmol/reaction), with a IC-specificdetection probe of SEQ ID NO:63 (0.75 pmol/reaction) for both assays.

TMA-based FluA and FluB assays with real-time detection resulted in 100%specificity for their respective influenza virus targets when testedagainst other respiratory viruses, microbes (bacteria and fungi)commonly associated with oral or respiratory flora or infections, andseveral human influenza virus subtypes. Tests performed on clinicalsamples that had been collected during influenza seasons over afour-year period demonstrated 100% agreement in sensitivity andspecificity when the TMA-based and PCR-based assays described hereinwere compared. TMA-based assays with real-time detection wereadvantageous positive detection results that indicated the presence ofthe influenza virus in the sample were obtained in a shorter time thanrequired for the real-time PCR-based assay to detect the viral nucleicacids. For example, TMA-based assays with real-time detection generallyprovided positive results in less than 45 min from the start of theamplification reaction, whereas PCR-based assays with real-timedetection typically required up to two hours or more to provide positiveresults.

Unless otherwise specified, reagents commonly used in the TMA-basedassays described herein include the following. Sample transport reagent:110 mM lithium lauryl sulfate (LLS), 15 mM NaH₂PO₄, 15 mM Na₂HPO₄, 1 mMEDTA, 1 mM EGTA, pH 6.7. Lysis buffer: 790 mM HEPES, 230 mM succinicacid, 10% (w/v) LLS, and 680 mM LiOH monohydrate. Target Capture Reagent(TCR): 250 mM HEPES, 1.88 M LiCl, 310 mM LiOH, 100 mM EDTA, pH 6.4, and250 μg/ml of paramagnetic particles (0.7-1.05μ particles, Sera-Mag™MG-CM) with (dT)₁₄ oligomers covalently bound thereto. Wash Solution: 10mM HEPES, 150 mM NaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02%(w/v) methylparaben, 0.01% (w/v) propylparaben, and 0.1% (w/v) sodiumlauryl sulfate, pH 7.5. Amplification reagent: a concentrated solutioncontaining 125 mM HEPES, 26.7 mM rATP, 33.3 mM rGTP, 5 mM each of rCTPand UTP, 1.33 mM each of dATP, dCTP, dGTP and dTTP, 8% (w/v) trehalose,pH 7.7, to which primers and probes may be added. TMA Enzymes: perreaction about 90 U/μl of MMLV reverse transcriptase (RT) and about 20U/μl of T7 RNA polymerase per reaction (where 1 U of RT incorporates 1nmol of dTTP in 10 min at 37° C. using 200-400 μM oligo-dT-primedpolyA-template, and 1 U of T7 RNA polymerase incorporates 1 nmol of ATPinto RNA in 1 hr at 37° C. using a T7 promoter in DNA template). ProbeReagent for AE-labeled probes: a solution of (a) 100 mM Li-succinate, 3%(w/v) LLS, 10 mM mercaptoethanesulfonate (MES), and 3% (w/v)polyvinylpyrrolidon, or (b) 100 mM Li-succinate, 0.1% (w/v) LLS, and 10mM MES. Hybridization Reagent: (C-type) 100 mM succinic acid, 2% (w/v)LLS, 100 mM LiOH, 15 mM aldrithiol-2, 1.2 M LiCl, 20 mM EDTA, and 3.0%(v/v) ethanol, pH 4.7. Selection Reagent: 600 mM boric acid, 182.5 mMNaOH, 1% (v/v) octoxynol (TRITON® X-100), pH 8.5 to 9.2, to hydrolyze AElabels on unbound oligomers. Detection Reagents for AE labels are DetectReagent I: 1 mM nitric acid and 32 mM H₂O₂, and Detect Reagent II: 1.5 MNaOH (see U.S. Pat. Nos. 5,283,174, 5,656,744, and 5,658,737).

EXAMPLE 1 TMA-Based Assays For Amplification and Detection of InfluenzaViruses

This example demonstrates a TMA-based assay for influenza virus type Adetection (FluA assay) and a TMA-based assay for influenza virus type Bdetection (FluB assay), both using an internal control (IC) that isamplified and detected in the same assay conditions.

The TMA-based FluA assay used primers of SEQ ID NO:8 (9 pmol/reaction)and SEQ ID NO:21 (15 pmol/reaction) and a fluorophore-labeled probe ofSEQ ID NO:29 (0.32 pmol/reaction) in a TMA reaction performedsubstantially as described above using varying amounts of influenzavirus A target (RNA transcript of SEQ ID NO:1, at 0 to 6.7 log copiesper reaction). Reactions were performed with or without IC components(primers of SEQ ID Nos. 61 and 62, probe of SEQ ID NO:63, and IC targetRNA transcripts of SEQ ID NO:65 at 0 or 2.3 log copies per reaction).Six replicates of each reaction were performed, each reaction in a wellof a standard 96-well plate, using 30 μl of amplification reagentcontaining the appropriate target oligonucleotides, incubated at 60° C.for 10 min and at 4° C. for 5 min, and then TMA enzymes were added toeach reaction in enzyme reagent (10 μl per reaction), reaction weremixed (30 sec vortex), followed by amplification incubation for 45-60min at 42° C. during which the fluorescent probe signal was detected attime intervals as described above (in a CHROMO4® instrument). Results ofthe tests are shown in Table 3, expressed as average time of emergenceof signal for the influenza virus A analyte (with standard deviation(SD) where calculated). Negative control samples (no influenza virus Atarget included) provided the background noise signal for calculation ofthe emergence times of the positive samples. The results show that thedynamic range of the assay for detection of the influenza virus A targetwas from 1.7 to 6.7 log copies of the viral target, and the IC wasdetected over the viral target titration range (up to 6.7 log copies).

TABLE 3 Real-Time Detection of Influenza Virus A During In Vitro NucleicAcid Amplification Influenza virus A target Average Emergence Time ± SD(min) (log copies) With IC (2.3 log copies) Without IC 1.70 31.5 24.2 ±0.51 2.70 28.9 ± 1.34 21.3 ± 0.38 3.70 25.6 ± 1.04 19.0 ± 0.45 4.70 19.8± 0.33 17.0 ± 0.07 5.70 16.6 ± 0.65 15.2 ± 0.18 6.70 14.6 ± 0.23 13.5 ±0.26

Separate TMA-based assays were performed using substantially the sameprocedure described above, but with 2.7 or 3.7 log copies of influenzavirus A target (transcripts of SEQ ID NO:1) amplified by using differentpromoter primers (SEQ ID Nos. 8, 9, 10 and 12, each tested separately at9 pmol/reaction) with a second primer of SEQ ID NO:21 (15pmol/reaction), and amplicons detected by using a fluorophore-labeledprobe of SEQ ID NO:29 (8 pmol/reaction). Similar results for positivesignals were obtained in these tests using the different combinations ofprimers although some combinations were more efficient at amplificationas determined by the shorter times of signal emergence. For primers ofSEQ ID Nos. 8 and 21, the emergence times were 20.9 to 25.8 min, forprimers of SEQ ID Nos. 9 and 21, the emergence times were 30.4 to 34.8min, for the primers of SEQ ID Nos. 10 and 21, the emergence times were19.4 to 27.9 min, and for the primers of SEQ ID Nos. 12 and 21, theemergence times were 19.8 to 25.8 min.

TMA-based FluB assays were performed using similar conditions butinfluenza virus B-specific primers and probes. In one embodiment,primers of SEQ ID NO:39 (9 pmol/reaction) and SEQ ID NO:44 (15pmol/reaction) were used to amplify varying amounts of the influenzavirus B target (RNA transcript of SEQ ID NO:32, at 0 to 7.7 log copiesper reaction), and amplicons were detected using fluorophore-labeledprobe of SEQ ID NO:52 (8 pmol/reaction). Reactions were performedsubstantially as described above, with or without IC components (primersof SEQ ID Nos. 64 and 62, probe of SEQ ID NO:63, and IC targets thatwere RNA transcripts of SEQ ID NO:65 at 0 or 2.3 log copies perreaction). Results of these FluB assays are shown in Table 4, expressedas average time of signal emergence for the influenza virus B analyte(with standard deviation (SD) where calculated). Results of negativecontrol samples (no influenza virus target) served as the backgroundnoise signal for calculation of the emergence times of the positivesamples. These results show that the dynamic range of the FluB assay wasfrom 1.7 to 7.7 log copies of influenza virus B target and the IC wasdetected up to 5.7 log copies of the influenza virus B target perreaction.

TABLE 4 Real-Time Detection of Influenza Virus B Nucleic During In VitroNucleic Acid Amplification Influenza virus B target Average EmergenceTime ± SD (min) (log copies) With IC (2.3 log copies) Without IC 1.7038.8 ± 0.37 41.0 ± 2.62 2.70 35.9 ± 0.27 36.1 ± 1.76 3.70 31.7 ± 1.7031.8 ± 1.26 4.70 27.6 ± 1.07 28.6 ± 0.63 5.70 24.5 ± 0.85 25.5 ± 0.276.70 20.6 ± 0.12 21.0 ± 0.73 7.7 17.2 ± 0.19 16.9 ± 0.18

Separate TMA-based FluB assays were performed using conditions likethose described above, but detected with different fluorophore-labeledprobes of SEQ ID Nos. 52, 55 and 58, which provided similar results. Inthese TMA-based FluB assays, the primers were to SEQ ID NO:39 (3pmol/reaction) and SEQ ID NO:44 (15 pmol/reaction) to amplify theinfluenza virus B RNA target at 8 log copies per reaction. Each of thedetection probes (about 0.3 pmol/reaction) were tested separately inseven replicate amplification and detection assays as described above.For these assays, the average emergence times were: 14.0±0.16 min forthe SEQ ID NO:52 probe, 10.7±0.22 min for the SEQ ID NO:55 probe, and12.0±0.33 min for the SEQ ID NO:58 probe. In separate experimentsperformed using similar conditions but with fluorophore-labeled probesof SEQ ID Nos. 51, 52, 53, 56, and 57, and lower amounts of theinfluenza virus B target RNA (4.3 log copies per reaction), the averageemergence times were: 24.9±0.12 min for the SEQ ID NO:56 probe,25.8±0.05 min for the SEQ ID NO:57 probe, 26.5±0.30 min for the SEQ IDNO:52 probe, 26.6±0.59 min for the SEQ ID NO:53 probe, and 31.8±0.48 minfor the SEQ ID NO:51 probe.

In similar tests, the TMA-based FluA and FluB assays were performedusing different combinations of IC primers. In the FluA assays,influenza virus A-specific primers of SEQ ID Nos. 9 and 21 amplified theinfluenza virus A RNA (transcript of SEQ ID NO:1 at 3.7 log copies perreaction), and amplicons were detected using a fluorophore-labeledinfluenza virus A-specific probe of SEQ ID NO:29. In the FluB assays,influenza virus B-specific primers of SEQ ID Nos. 39 and 44 amplifiedthe influenza virus B RNA (transcripts of SEQ ID NO:32 at 4.7 log copiesper reaction), and amplicons were detected using a fluorophore-labeledprobe of SEQ ID NO:48. For both the FluA and FluB assays, differentcombinations of IC-specific oligomers were used to amplify the IC (RNAtranscript of SEQ ID NO:65 at 2.3 log copies per reaction): primer ofSEQ ID NO:62 (10 pmol/reaction) was combined with about 0.5pmol/reaction of primer of SEQ ID Nos. 61, 64, 66 or 67, and then the ICamplicons were detected in all reactions by using a fluorophore-labeledprobe of SEQ ID NO:63. The results of these tests showed that all of thecombinations of IC-specific primers could be used in the FluA and FluBassays without interfering with detection of the respective influenzaviral target for the assay, but the promoter primer of SEQ ID NO:61 wasoptimal for the FluA assay whereas the promoter primer of SEQ ID NO:64was optimal for the FluB assay.

EXAMPLE 2 Specificity of TMA-Based FluA and FluB Assays

This example demonstrates the specificity of the TMA-based FluA and FluBassays, which specifically detected the intended viral target for eachtest and did not provide a positive signal when samples contained otherbacterial or viral agents commonly found in normal human oral flora orrespiratory infections. The TMA-based FluA and FluB assays wereperformed substantially as described in Example 1 except that the testedsamples included known microbes or viruses and did not contain influenzavirus A or influenza virus B nucleic acid (except for positivecontrols). That is, the assays provided negative results showing thatthe assays did not cross-react with other microbial or viral nucleicacids. An IC RNA was included in all of the tests to demonstrate thatthe assay conditions and amplification and detection steps had beenperformed appropriately to detect the IC target (or any cross-reactivetarget) in the sample.

Each sample containing a known virus was tested independently using theTMA-based FluA test with IC and the FluB test with the same IC. SeparateFluA and FluB assays were performed simultaneously under the sameconditions using positive control samples that contained influenza virusA or influenza virus B targets. Positive controls included four sourcesof influenza virus A nucleic acid and two sources of influenza virus Bnucleic acid, each tested individually at 10⁵ and 10² copies perreaction (with American Type Culture Collection (ATCC) accession numbersprovided below). Positive control samples for influenza virus Aincluded: isolates of A/Port Chalmers/1/73(H3N2) (ATCC VR-810),A/Mal/302/54(H1N1) (ATCC VR-98), and A/Hong Kong/8/68(H3N2) (ATCCVR-544), and in vitro RNA transcripts of Flu A/Beijing(H1N1) (an isolatefrom the Center for Disease Control (CDC), Atlanta, Ga.). Positivecontrol samples for influenza virus B included: in vitro RNA transcriptsof B/Maryland/1/59 (ATCC VR-296) and isolate B/Lee/40 (ATCC VR-101).

The TMA-based FluA assay was performed by using primers of SEQ ID NO:8(9 pmol/reaction) and SEQ ID NO:21 with LNA for residues 1 to 3 (15pmol/reaction), and a molecular beacon probe of SEQ ID NO:29 (0.267pmol/reaction) for real-time detection of the TMA amplicons. Thereactions included an IC (RNA transcripts of SEQ ID NO:65 at 200copies/reaction) that was amplified by using primers of SEQ ID Nos. 61and 62 (0.5 and 15 pmol/reaction, respectively), and the IC ampliconswere detected in real time by using a molecular beacon probe of SEQ IDNO:63 (0.6 pmol/reaction). Additional positive controls were tested atthe same time using the same conditions but using samples that containedknown amounts of an influenza virus A target (RNA transcripts of SEQ IDNO:1 at 2 or 5 log copies per reaction).

The TMA-based FluB assay was performed by using primers of SEQ ID NO:39(9 pmol/reaction) and SEQ ID NO:44 with LNA for residues 1 to 3 (15pmol/reaction), and a molecular beacon probe of SEQ ID NO:52 (0.267-0.32pmol/reaction) for real-time detection of TMA amplicons. The assaysincluded an IC (RNA transcripts of SEQ ID NO:65 at 200 copies/reaction)that was amplified by using primers of SEQ ID Nos. 64 and 62 (0.5 and 15pmol/reaction, respectively), and IC amplicons were detected in realtime by using a molecular beacon probe of SEQ ID NO:63 (0.6pmol/reaction). Positive controls were tested at the same time under thesame conditions but using samples that contained a known amount of theinfluenza virus B target (RNA transcripts of SEQ ID NO:32 at 2 or 5 logcopies per reaction).

The TMA-based FluA assay gave positive results for all tested samplesthat contained influenza virus A nucleic acids (average emergence timefor positive signals was 13.9 min) and negative results for all controlsamples that contained influenza virus B nucleic acid. Similarly, theTMA-based FluB assay gave positive results for all tested samples thatcontained influenza virus B nucleic acids (average emergence time forpositive signals was 21.9 min) and negative results for all influenzavirus A control samples.

The common normal or pathogenic microbe species and isolates (with ATCCaccession Nos.) that were tested in samples included: Bordetellabronchiseptica (ATCC 10580), Bordetella pertussis (ATCC 8467),Bordetella parapertussis (ATCC 15311), Burkholdia cepacia (clinicalisolate), Candida albicans (ATCC 18804), Corynebacterium striatum (ATCC6940), Escherichia coli (ATCC 29214), Enterococcus faecalis (ATCC19433), Fluoribacter bozemanii (ATCC 33217), Fluoribacter dumoffii (ATCC33279), Haemophilus influenzae (ATCC 33391), Haemophilus parainfluenzae(ATCC 7901), Klebsiella pneumoniae (ATCC 23357), Legionella longbeacheae(ATCC 33484), Legionella pneumophila subsp. pneumophila (type 3, ATCC33155; type 4, ATCC 33156; type 6, ATCC 33215; and type 11, ATCC 43130),Legionella pneumophila subsp. fraseri (type 5, ATCC 33216), Moraxellacattarhalis (ATCC 25238), Pseudomonas aeruginosa (ATCC 27853 and ATCC9027), Proteus mirabilis (ATCC 25933), Staphylococcus aureus (ATCC25923), Staphylococcus epidermidis (ATCC 14990), Streptococcusagalactiae (GBS) (ATCC 13813), Streptococcus gordonii (viridans strep)(ATCC 33399), Streptococcus mutans (ATCC 25175), Streptococcus oralis(ATCC 10557), Streptococcus pneumoniae (ATCC 35088), Streptococcuspyogenes (GAS) (ATCC 12344), and Tatlockia micdadiae (ATCC 33204).Isolates were grown on appropriate media and then a 1 μl loop of cellswas added to a microcentrifuge tube containing 150 μl of lysis reagent(a succinate buffered detergent solution), vortexed, and incubated at95° C. for 10 min to lyse cells. Lysates were stored frozen (−20° C.)until tested, when the lysate was thawed and diluted (1:100) into waterbefore use in the assay. For FluA and FluB assays performed using themicrobial lysates, no positive responses were obtained for any of thelysates, but positive responses were detected for the internal control(IC) indicating that the assays were preformed properly with theappropriate reagents that did not cross-react with nucleic acids presentin the microbial lysates. Positive controls (containing influenza virusA or B nucleic acids) gave positive results only for the intended targetof the respective assay, thus demonstrating the specificity of theTMA-based FluA and FluB assays for their intended viral targets.

Assays were similarly performed to test for cross-reactivity of theTMA-based FluA or FluB assay with known non-influenza human viruses thatare potential viral respiratory pathogens. Samples containing thefollowing viruses (with ATCC accession numbers) were tested: Adenovirus1 (strain Adenoid 71, ATCC VR-1), Adenovirus 4 (strain R1-67, ATCCVR-4), Adenovirus 7 (strain Gomen, ATCC VR-7), Adenovirus 11 (strainSlobitski, ATCC VR-12), Adenovirus 18 (strain DC, ATCC VR-19),Adenovirus 29 (strain BP-6, ATCC VR-272), coronoavirus 229E (Group 1type, ATCC VR-740), coronavirus OC43 (Group 2 type, ATCC VR-759),parainfluenza virus type 1 (a clinical isolate and ATCC VR-1380),parainfluenza virus type 2 (a clinical isolate and strain Greer, ATCCVR-92), parainfluenza type 3 (clinical isolate), parainfluenza 4a(strain M-25, ATCC VR-1378), rhinovirus (clinical isolate), andRespiratory Syncyntial Viruses (RSV) (a clinical isolate; strain BWV/14617/85, ATCC VR-1400; and strain A-2, ATCC VR-1540). A total of 42viral samples were tested, which included those listed above andseparate clinical isolates collected over four years of influenzaseasons. Viral RNA was extracted using a standard protocol that collectsnucleic acids non-specifically on a support, washes the collectednucleic acids, and elutes the nucleic acids from the support into anaqueous solution (QIAAMP™ Viral RNA Mini Vacuum Protocol, Qiagen Inc.).All viral nucleic acids were tested immediately after extraction orstored frozen (−70 to −80° C.) until tested. For all of thenon-influenza virus nucleic acids tested, both the FluA and the FluBassays gave negative results for the non-influenza viral nucleic acidsbut gave positive results for the IC, indicating that the assays wereperformed properly with appropriate components that did not cross-reactwith the non-influenza viral nucleic acids. All of the positive controlsthat contained influenza virus target nucleic acid gave the appropriatepositive responses for the intended target specific for the FluA andFlub assays. That is, the FluA assay detected influenza virus A but notinfluenza virus B, and the FluB assay detected influenza virus B but notinfluenza virus A. These results show that the FluA and FluB assays arespecific for their intended influenza viral targets and do notcross-react with other human viral targets that are potentially found inrespiratory samples.

EXAMPLE 3 Comparison of TMA-Based and PCR-Based Assays for InfluenzaVirus Detection

This example describes tests that compared the time for positivedetection of influenza virus A and influenza virus B by usingtype-specific tests based on TMA and PCR amplification methods. Theresults show that TMA-based assays provided detection results soonerthan obtained with assays based on real-time RT-PCR methods.

TMA-based FluA assays were performed substantially as described inExample 1, using primers of SEQ ID NO:9 (3 pmol/reaction) and SEQ IDNO:21 (15 pmol/reaction) to amplify target RNA transcripts of SEQ IDNO:1 (1 to 7 log copies per reaction), and detecting the amplicons byusing a fluorophore-labeled probe of SEQ ID NO:29 (8 pmol/reaction). Theprimer of SEQ ID NO:21 was tested separately in two versions: one thatwas completely DNA, and one that was DNA except for LNA at residues 1 to3. Six replicate assays were performed for each condition. The TMA-basedFluB assay was performed substantially as described in Example 1, usingprimers of SEQ ID NO:39 (3 pmol/reaction) and SEQ ID NO:44 to amplifytarget RNA transcripts of SEQ ID NO:32 (1 to 7 log copies per reaction),and detecting the amplicons by using a fluorophore-labeled probe of SEQID NO:52 (8 pmol/reaction). Six replicate assays were performed for eachcondition.

Real-time RT-PCR assays for FluA were performed substantially asdescribed above using primers of SEQ ID Nos. 68 and 69 to amplify thesame RNA target used in the TMA-based FluA assay, andfluorophore-labeled probe of SEQ ID NO:70 to detect the PCRamplification products. Real-time RT-PCR assays for FluB were performedsubstantially as described above using primers of SEQ ID Nos. 71 and 72to amplify the same RNA target used in the TMA-based FluB assay, and afluorophore-labeled probe of SEQ ID NO:73 to detect the PCRamplification products. Each 50 μl reaction mixture contained includestandard reagents (TAQMAN® One-Step RT-PCR Master Mix Reagents Kit,Applied Biosystems, Inc., Foster City, Calif.) and used conditions assuggested by the supplier, with the amplification and detection stepsperformed using a thermocycler and fluorometer device (OPTICON™ 2 systemor CHROMO4™ Real-Time PCR Detector, Bio-Rad Laboratories, Inc.,Hercules, Calif.).

The results of the comparative tests are shown in Table 5 for the FluAassays and Table 6 for the FluB assays. The results are shown as average(mean) emergence time for positive signals (±standard deviation, whencalculated), determined from the initiation of the amplificationreactions. The results show that, for both FluA and FluB targets,TMA-based assays provide a positive response before a positive responsewas seen for the PCR-based assays for the same number of targets.Results in Table 5 show that TMA-based FluA assays that used anLNA-containing primer had improved amplification kinetics compared toTMA-based assays that used a complete DNA primer, particularly for teststhat contained fewer targets.

TABLE 5 Detection of Influenza Virus A Influenza virus A AverageEmergence Time ± SD (min) target TMA TMA (log copies) (SEQ 21 DNA) (SEQ21 LNA 1-3) PCR 1.00 32.5 ± 2.36 29.2 ± 1.91 55.0 ± 4.34 2.00 28.8 ±2.09 25.3 ± 1.22 58.1 ± 0.45 3.00 28.8 ± 1.31 21.8 ± 0.20 55.0 ± 0.444.00 25.6 ± 0.38 19.8 ± 0.64 49.2 ± 0.37 5.00 23.2 ± 0.19 17.8 ± 0.1844.6 ± 0.01 6.00 20.8 ± 0.10 15.7 ± 0.64 39.2 ± 0.41 7.00 18.7 ± 0.6514.2 ± 0.29 33.8 ± 0.12

TABLE 6 Detection of Influenza Virus B Influenza virus B target AverageEmergence Time ± SD (min) (log copies) TMA PCR 1.00 35.8 52.3 ± 0.142.00 36.0 ± 0.36 53.7 ± 0.52 3.00 32.7 ± 0.71 45.6 ± 0.14 4.00 29.8 ±0.66 41.0 ± 0.07 5.00 25.7 ± 0.63 35.6 ± 0.06 6.00 22.4 ± 0.57 30.6 ±0.13 7.00 19.2 ± 0.22 25.8 ± 0.07

In separate experiments, TMA-based FluB assays were performed similarlybut using in separate reactions a promoter primer of SEQ ID NO:39 (3pmol/reaction) combined with different synthetic versions of SEQ IDNO:44 primer (completely DNA, or DNA with LNA at positions 1 to 3, orDNA with LNA at positions 1 to 4, all used at 15 pmol/reaction). Thereactions amplified FluB RNA transcript targets at 2.7 and 3.7 logcopies per reaction and the amplicons were detected by using afluorophore-labeled probe of SEQ ID NO:52. Results of these assaysshowed that the LNA-containing SEQ ID NO:44 primers improvedamplification and assay kinetics compared to the completely DNA primerof SEQ ID NO:44.

EXAMPLE 4 Detection of Influenza Virus in Clinical Samples

This example shows that TMA-based FluA and FluB assays that detect theamplicons in real time provided positive results for samples thatcontain the target influenza virus sooner than for real-timeRT-PCR-based assays performed on the same nineteen clinical samples.Assays were performed substantially as described in Example 3, but usingan aliquot of prepared clinical sample nucleic acid in place of thetarget influenza virus RNA transcripts. Some of the TMA-based assaysincluded an IC, substantially as described in Example 2. All assaysincluded a negative control (no target) and positive controls (2 or 5log copies of the intended target influenza virus RNA transcripts). Allnegative controls gave negative results.

The TMA-based FluA assays were performed substantially as describedabove but using an aliquot of prepared clinical sample nucleic acid inplace of the FluA RNA transcripts as target. The assays used primers ofSEQ ID Nos. 8 and 21, and a fluorophore-labeled probe of SEQ ID NO:29.The TMA-based assays performed with an IC included the IC target (200copies of RNA transcript of SEQ ID NO:65), IC-specific primers of SEQ IDNos. 61 and 62, and the IC-specific probe of SEQ ID NO:63. The RT-PCRFluA assays were performed substantially as described above but using analiquot of prepared clinical sample nucleic acid in place of the FluARNA transcripts as target. The RT-PCR FluA assays used primers of SEQ IDNos. 68 and 69 and probe of SEQ ID NO:70 during 55 cycles of PCRamplification.

The TMA-based FluB assays were performed as described above but using analiquot of prepared clinical sample nucleic acid in place of the FluBRNA transcripts as target. The assays used primers of SEQ ID Nos. 39 and44, and a fluorophore-labeled probe of SEQ ID NO:48. The TMA-basedassays performed with an IC included the IC target (200 copies of RNAtranscript of SEQ ID NO:65), using IC-specific primers of SEQ ID Nos. 64and 62, and the IC-specific probe of SEQ ID NO:63. The RT-PCR FluBassays were performed substantially as describe above but using analiquot of prepared clinical sample nucleic acid in place of the FluBRNA transcripts as target. The RT-PCR FluB assays used primers of SEQ IDNos. 71 and 72 and probe of SEQ ID NO:73 during 55 cycles of PCRamplification

The FluA clinical samples were provided from a clinical testinglaboratory (Warde Medical Center) and included an isolate ofA/Beijing(H1N1) and other specimens or isolates obtained from patientsduring multiple influenza seasons. The FluB clinical samples representedsix different isolates or specimens. Clinical samples were treatedbefore amplification by purifying the nucleic acids in the samples usinga non-specific method (QiAmp RNA extraction kits, using the supplier'srecommended conditions). A 5 μl aliquot of the eluate containing nucleicacids isolated from the clinical samples was used per reaction.

The results of the FluA tests are shown in Table 7, and those of theFluB tests are shown in Table 8. The results in Table 7 show that theTMA-based and PCR-based assays provided consistent results for positiveor negative specimens, and that the emergence times for the positivesamples in the TMA-based assays were consistently earlier than for thesame samples tested in the PCR-based assays, except for sample A1 testedusing the TMA-based assay that included an IC. The negative specimen(A11) gave a positive result for the IC in the TMA-based assay,indicating that the result was a true negative for influenza virus A.The results in Table 8 similarly show that the TMA-based and PCR-basedassays provided consistent positive results for the specimens but thatthe emergence times for the positive TMA-based assays were sooner thanfor the PCR-based assays. The total time required to perform thecomplete assay, i.e., not just emergence time of signal detected in theamplification reaction, was shorter for the TMA-based assays for bothFluA and FluB (about 45 min) compared to PCR-based assays for the sametargets (about 120 min).

TABLE 7 Detection of Influenza Virus A Amplified Nucleic Acid EmergenceTime (min) Specimen TMA without IC TMA with IC PCR without IC A1 16.937.0 35.4 A2 14.5 15.5 36.5 A3 (A/Beijing 12.8 13.5 32.5 (H1N1)) A4 13.313.8 26.2 A5 12.5 13.7 25.8 A6 15.0 15.7 31.3 A7 15.1 16.3 33.5 A8 14.215.8 29.1 A9 12.6 14.1 22.9 A10 28.3 29.8 40.5 A11 negative negativenegative (IC positive) A12 14.0 14.8 30.1 A13 14.9 15.7 27.6 A14 11.111.6 24.5 A15 12.1 12.7 28.4 A16 11.6 12.8 29.3 A17 13.6 15.0 27.6 A1811.1 12.1 30.0 19 13.0 14.2 33.5 Positive control 20.9 35.7 37.7 (10²copies) Positive control 15.3 16.5 32.4 (10⁵ copies)

TABLE 8 Detection of Influenza Virus B Amplified Nucleic Acid EmergenceTime (min) Specimen TMA without IC TMA with IC PCR without IC B1 13.814.4 27.8 B2 13.8 16.3 26.3 B3 16.2 20.3 30.8 B4 17.8 23.3 30.9 B5 21.129.4 33.6 B6 13.6 16.2 27.6 Positive control 24.5 24.6 50.4 (10² copies)Positive control 16.5 16.8 Not tested (10.sup.5 copies)

EXAMPLE 5 Detection of Amplified Sequences of Influenza Viruses by UsingChemiluminescent Probes

This example demonstrates assays that involved transcription associatedamplification of influenza virus A or influenza virus B nucleic acidsfollowed by detection of the amplification products by using AE-labeledprobes specific for the influenza virus A or influenza virus B amplifiedsequences. The probes hybridized to the amplicons emit chemiluminescentsignals that are detected in a homogeneous assay format.

For TMA-based FluA assays, standard TMA reactions were performed toamplify sequences in 500,000 copies of the FluA target RNA (transcriptof SEQ ID NO:1) by using different combinations of two primers (SEQ IDNo. 7, 9, or 11 combined with SEQ ID No. 21, 22, or 24). Followingamplification, hybridization to a probe of SEQ ID NO:26 labeled with AEbetween residues 7 and 8 was used to detect amplicons produced in theTMA reaction. Negative controls were assays performed identically but inreaction mixtures that contained no FluA target RNA. Followingamplification, the amplicons were hybridized to the AE-labeled probe inhybridization reagent at 60° C. for 30 min and then cooled to roomtemperature for 5 min. Then, AE labels in unbound probes wereselectively hydrolyzed by using selection reagent and incubation at 60°C. for 10 min, followed by room temperature for 15 min. Chemiluminescentsignals were elicited by using detect reagent I, followed byneutralization with detect reagent II, and chemiluminescence wasdetected on a luminometer (LEADER® HC, Gen-Probe Incorporated) asrelative light units (RLU)) substantially as described previously (U.S.Pat. Nos. 5,283,174 and 5,656,744, Arnold et al., and U.S. Pat. No.5,658,737, Nelson et al., at column 25, lines 27-46; Nelson et al.,1996, Biochem. 35:8429-8438 at 8432). The results of the assays areshown in Table 9. The results show that a homogeneous chemiluminescentdetection system also detects influenza virus A nucleic acid followingtranscription associated amplification by using different combinationsof FluA-specific amplification oligomers.

TABLE 9 Detection of Influenza Virus A Amplified Nucleic Acid PrimersRLU for FluA RLU for (SEQ ID NOs) Positive Sample Negative Control  7and 21 4.92 × 10.sup.6 2.76 × 10.sup.3  9 and 21 4.83 × 10.sup.6 2.74 ×10.sup.3 11 and 21 4.00 × 10.sup.6 2.80 × 10.sup.3  7 and 22 4.84 ×10.sup.6 2.95 × 10.sup.3  9 and 22 3.56 × 10.sup.6 2.65 × 10.sup.3 11and 22 4.25 × 10.sup.6 2.74 × 10.sup.3  7 and 24 3.82 × 10.sup.6 2.69 ×10.sup.3  9 and 24 2.35 × 10.sup.6 2.74 × 10.sup.3 11 and 24 1.48 ×10.sup.6 2.56 × 10.sup.3

Probes specific for FluB target sequences were similarly shown tospecifically detect influenza virus B sequences. In these assays, asynthetic RNA target (SEQ ID NO:33, at 2 pmol/reaction) was mixed withAE-labeled probes of SEQ ID NO:48 (labeled between residues 8 and 9),SEQ ID NO:49 (labeled between residues 9 and 10), and SEQ ID NO:50(labeled between residues 7 and 8) in a mixture that mimics a TMAreaction mixture, and then the detection step was performedsubstantially as described above. That is, detection of the FluB targetsequences was performed directly without amplification from an initialRNA target. The average results of these assays (n=5 for each condition)are shown in Table 10. These results show that influenza virus B targetsequences are detected by using AE-labeled probes in conditions similarto those that result from amplification in a TMA-based assay.

TABLE 10 Detection of Influenza Virus B Amplified Nucleic Acid FluB AE-RLU for FluB RLU for labeled Probe Positive Samples Negative ControlsSEQ ID NO: 49 6.91 × 10.sup.5 1.53 × 10.sup.3 SEQ ID NO: 50 2.18 ×10.sup.5 1.16 × 10.sup.3 SEQ ID NO: 48 2.10 × 10.sup.6 8.91 × 10.sup.2

EXAMPLE 6 TMA-Based Assays for Influenza Virus Detection

This example shows that TMA-based FluA and FluB assays give comparableresults for influenza virus detection when performed manually or byusing an automated device. For both FluA and FluB assays, the methodsteps were performed using identical conditions but each assay containedthe target-specific oligomers for the respective target of the assay,influenza virus A or influenza virus B.

Samples were prepared containing synthetic targets for the respectiveassays (RNA transcripts of SEQ ID NO:1 for FluA assays, or RNAtranscripts of SEQ ID NO:32 for FluB assays, at 0, 250, 500, 1000 or5000 copies/ml) and mixed with target capture reagent (TCR) in a finalvolume of 0.5 ml containing a target capture probe (200 pmol/ml)specific for the respective influenza virus target (SEQ ID NO:6 for FluAassays, and SEQ ID NO:35 for FluB assays). Target capture was performedby incubating the mixture containing the target RNA, capture probe andTCR at 62° C. for 30, and then at room temperature for 30 min. Magneticparticles with captured influenza virus nucleic acids were separated toan inner portion of the container by using a magnetic field, and thesample liquid was removed. Captured target nucleic acids on theparticles were washed with 1 ml/reaction of Wash Solution, particleswere separated magnetically, and the Wash Solution was removed. Washedparticles with captured influenza viral target nucleic acids weresuspended in 75 μl of a nucleic acid amplification reagent for TMAcontaining the appropriate amplification oligomers (SEQ ID NO:10 at 300pmol/ml, and SEQ ID NO:21 with LNA at residues 1 to 3 at 500 pmol/ml forFluA assays; SEQ ID NO:39 at 300 pmol/ml, and SEQ ID NO:44 with LNA atresidues 1 to 3 at 500 pmol/ml for FluB assays). TMA reactions wereincubated at 60° C. for 10 min, then at 42° C. for 5 min, and TMAenzymes were added, mixed, and incubation continued at 4° C. for 60 min.Detection was performed substantially as described in Example 5 usingAE-labeled probes specific for the intended target of the assay (SEQ IDNO:26 at 5×10⁷ RLU/ml for FluA assays, and SEQ ID NO:48 at 5×10⁷ RLU/mlfor FluB assays), and chemiluminescent signals produced in thehomogeneous assay format were detected and measured.

The FluA and FluB assays were performed separately using 10 replicatesfor each assay condition. The assay steps were performed manually or byusing an automated system (for details see U.S. Pat. Nos. 6,605,213 and6,846,456). The results of these assays are shown in FIG. 1 for FluAassays and FIG. 2 for FluB assays. FIG. 1 graphs the signals (averageRLU) detected in the TMA-based FluA assays for each tested amount ofinfluenza virus A target (250 to 5000 copies/ml) in assays performedmanually (light bars) or by using an automated system (dark bars). FIG.2 graphs the signals (average RLU) detected in TMA-based Flue assays foreach tested amount of influenza virus B target (250 to 5000 copies/ml)in assays performed manually (light bars) or by using an automatedsystem (dark bars). The results shown in FIGS. 1 and 2 illustrate thatboth TMA-based assays detected 250 copies/ml or greater of therespective intended target influenza virus nucleic acids, whether thesteps were performed manually or in an automated system.

1. A method for amplifying and detecting influenza virus A in a samplecomprising the steps of: (a) contacting a sample suspected of containinginfluenza virus A nucleic acid with a combination of oligomers, saidcombination of oligomers comprising: (i) a first amplification oligomerhaving a target specific sequence consisting essentially of SEQ ID NO:23or the reverse complement of SEQ ID NO:23; and (ii) a secondamplification oligomer that is sufficiently complementary to theinfluenza A nucleic acid to hybridize and participate in a nucleic acidamplification reaction, the second amplification oligomer having atarget specific sequence 21 to 23 bases long, wherein the targetspecific sequence contains SEQ ID NO:17 or the reverse complement of SEQID NO:17; (b) performing a nucleic acid amplification reaction; and (c)detecting an amplification product generated in step (b), therebydetermining the presence or absence of influenza A virus in the sample.2. The method of claim 1, wherein the second amplification oligomer hasa target specific sequence selected from the group consisting of SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18 and the reverse complement of any one of the aforementionedsequences.
 3. The method of claim 1, wherein either the firstamplification oligomer or the second amplification oligomer contains apromoter sequence.
 4. The method of claim 1, wherein the detecting stepuses an oligonucleotide detection probe.
 5. The method of claim 4,wherein the contacting step further comprises contacting the sample withthe detection probe.
 6. The method of claim 4, wherein the detectionprobe is added to the reaction after step (a).
 7. The method of claim 4,wherein the target specific sequence of the detection probe is selectedfrom the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and the reversecomplement of any one of the aforementioned sequences.
 8. The method ofclaim 4, wherein the detection probe comprises at least one detectablelabel.
 9. The method of claim 8, wherein the detection probe is a TaqManprobe, a molecular torch or a molecular beacon.
 10. The method of claim8, wherein the detectable label is a chemiluminescent compound.
 11. Themethod of claim 1, wherein detecting step is a real-time detectionreaction.
 12. The method of claim 1, wherein the detecting step uses ahomogenous detection reaction.
 13. The method of claim 1, wherein thenucleic acid amplification reaction is a multiplex reaction.
 14. Themethod of claim 13, wherein the nucleic acid amplification reaction is amultiplex reaction comprising a target nucleic acid that is an internalcontrol target sequence.
 15. The method of claim 14, wherein theinternal control target sequence is amplified using one or moreamplification oligomers having a target specific sequence selected fromthe group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:67, and combinations thereof.
 16. The method of claim13, wherein the nucleic acid amplification reaction is a multiplexreaction and wherein the sample further contains a nucleic acid frominfluenza virus B.
 17. The method of claim 1 further comprising a targetcapture step to separate or purify the influenza virus A nucleic acidprior to the contacting step wherein the target capture step uses atarget capture probe.
 18. The method of claim 17, wherein the targetspecific sequence of the target capture probe is selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, reverse complement of SEQ IDNO:3, and the reverse complement of SEQ ID NO:4.
 19. The method of claim18, wherein the target capture probe is selected from the groupconsisting of SEQ ID NO:5 and SEQ ID NO:6.
 20. The method of claim 1,wherein the amplification reaction uses thermal cycling.
 21. The methodof claim 1, wherein the amplification reaction is isothermal.